Mehrez M. Jadaon

Transmission of a Klebsiella pneumoniae clone harbouring genes for CTX-M-15-like and SHV-112 enzymes in a neonatal intensive care unit of a Kuwaiti hospital

The spread of antibiotic-resistant bacteria has become a large problem in most countries including Kuwait. This antibiotic resistance is usually due to the production of extended-spectrum beta-lactamase (ESBL) enzymes such as SHV, TEM and CTX-M. This study reports the emergence and spread of an ESBL-producing Klebsiella pneumoniae clone in a neonatal intensive care unit (NICU) in a Kuwaiti hospital. Eight ESBL-producing K. pneumoniae isolates were from blood cultures of seven neonates, and two were from the fingers of two healthcare workers in a NICU in Al Jahra Hospital, Kuwait. All isolates were obtained in February-March 2006, except for one, which was obtained in August 2005. Identification of the bacteria was based on traditional bacteriological and biochemical tests using the Vitek system. Antibiotic susceptibility was tested by the disc diffusion method using 16 different antibiotics. ESBLs were detected using disc approximation and double-disc synergy methods and confirmed as ESBLs using Etest. PCR and DNA sequencing were performed to determine the genotypes and mutations in the beta-lactamase genes (blaTEM, blaSHV and blaCTX-M). Genetic relatedness was determined by PFGE. All isolates were confirmed to have ESBLs by the Vitek system, disc approximation test, double-disc diffusion test and Etest, being resistant to cefotaxime, ceftazidime, cefepime, gentamicin, tobramycin and ciprofloxacin but susceptible to tetracycline and trimethoprim-sulfamethoxazole. Molecular studies showed the isolates to have TEM-1 beta-lactamase, a CTX-M-15-like ESBL and the newly discovered SHV-112 ESBL. PFGE showed that all isolates had identical banding patterns. The results indicate that a single clone of ESBL-producing K. pneumoniae caused bloodstream infections among babies in a NICU of a Kuwaiti hospital, and may have emerged at least 5 years ago. This clone was also present on the hands of healthcare workers, suggesting that they may have been involved in its transmission. Further studies are recommended to determine whether this clone is also spreading in other Kuwaiti hospitals.

Factor V Leiden mutation in Arabs in Kuwait by real-time PCR: different values for different Arabs

Factor V Leiden (FVL) mutation (G1691A) is a risk factor for development of venous thromboembolic disorders. FVL was found mostly in Caucasians (1–15%) but was almost absent in non-Caucasians. Studies on Arab patients and populations revealed very inconsistent results. This study reports FVL in Arabs living in Kuwait with a focus on the nationality of the Arab subjects studied. Whole-blood samples were collected from 400 healthy Arabs who were 268 Kuwaitis (67%), 50 Syrians (12.5%), 34 Jordanians (8.5%), 8 Palestinians (2%) and 40 Egyptians (10%). DNA extraction was carried out for these blood samples and real-time PCR was performed to detect the presence of FVL. Generally, 36 cases (9%) had the mutation (33 were heterozygous and 3 were homozygous), with an allelic frequency of 0.049. The prevalence of FVL differed in different Arabic cases: Kuwaitis 4.5%, Egyptians 15%, Syrians 16%, Jordanians 23.5% and Palestinians 25%. The allelic frequency was 0.022 in the Kuwaitis and 0.088–0.132 in non-Kuwaitis. The three homozygous cases were from Syria, Jordan and Egypt. In conclusion, the prevalence of FVL in Arabs living in Kuwait is as high as in Caucasians. There is a difference in prevalence among Arabs themselves, being relatively lower in Kuwaitis than in non-Kuwaitis.

High prevalence of activated protein C resistance and factor V Leiden mutation in an Arab population and patients with venous thrombosis in Kuwait.

IntroductionActivated protein C resistance (APC-R) because of clotting factor V Leiden mutation (FVL; Arg506Gln; G1691A) is a risk factor for the development of venous thromboembolic disorders (VTE). APC-R/FVL was reported to be very high in White patients with VTE (15% to 65%) and healthy populations (1% to 15%), and to be very low or absent in non-White patients. Studies on Arab patients and populations were very inconsistent. This study reports APC-R and FVL in Arabs living in Kuwait. Materials and MethodsWhole venous blood samples were collected from 400 patients with VTE and 200 healthy controls, all of whom were of Arab ethnicity living in Kuwait. The samples were used to separate plasma for an APC-R test, and DNA extraction for polymerase chain reaction and restricted fragment length polymorphism were performed. APC-R was on an automated hemostasis analyzer, and values less than 2.0 were reported as APC-R. Polymerase chain reaction and restricted fragment length polymorphism tests were performed using standard methods, and the results were reported as normal wild-type homozygous GG, FVL homozygous AA, or FVL heterozygous GA. ResultsSixty-three out of 400 patients (15.75%) and 4 out of 200 healthy controls (2%) had APC-R and at least one copy of FVL. Fifty-one patients and 4 controls were heterozygous whereas only 12 patients were homozygous. ConclusionThe prevalence of APC-R and FVL is quite high in Arabs living in Kuwait, being comparable with the prevalence reported in Whites, although being toward the lowest values reported there.

The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait

BackgroundEscherichia coli O25b-B2-ST131 are considered virulent extra-intestinal pathogens causing serious clinical complications such as urinary tract infection and bacteraemia. Our main objectives in this study were to characterise the multi-drug resistant (MDR) isolates of this lineage in Kuwait, and to demonstrate whether reduced susceptibility is spread clonally.ResultsA subset of 83 (10%) non-duplicate and non-selective E. coli O25b-B2-ST131 out of 832 MDR E. coli was identified and collected. Minimum inhibitory concentrations of the isolates were determined and pulsed-field gel electrophoresis was used for typing.The majority (95.2%) of the 83 E. coli O25b-B2-ST131 harboured at least one bla gene with blaCTX-M-15 being the most prevalent. blaCTX-M-2 was present in one isolate. Also one isolate harboured blaCTX-M-56, qnr B1 and blaCMY-2 genes and carried IncF1 plasmids of about 97kb and160 kb. qnr B and qnr S were found in 8 other blaCTX-M-15 containing isolates. The blaNDM, blaIMP, blaVIM and qnr A were not detected, however, the blaOXA-48 was present in two (2.4%).ConclusionsThe majority of isolates harbouring qnr genes demonstrated relatedness (≥85%) by PFGE. However, the diversity in PFGE profiles for the other MDR isolates reflected the changes in population genetics of E. coli O25b-B2-ST131. We identified for the first time the appearance of blaCTX-M-2 in the Middle East and blaCTX-M-56 outside the Latin American countries. The isolate harbouring blaCTX-M-56 also contained qnr B1 and blaCMY-2 genes and carried IncF1 plasmids. The appearance of a highly virulent E. coli O25b-ST131 that is resistant to penicillins, most cephalosproins, β-lactamase inhibitors as well as fluoroquinolones is a cause for concern.

Smoking, von Willebrand factor and ADAMTS-13 in healthy males.

While von Willebrand factor (vWF) has been reported to be elevated in smokers, there are no reports on the effects of smoking on its cleaving protease ADAMTS-13, particularly in subjects of Arab ethnicity. This study was conducted to determine the effects of smoking on vWF and ADAMTS-13 antigen and activity levels in Arab males. Venous blood samples from 80 smoking (at rest) and 80 non-smoking healthy males were collected after asking subjects to fast and refrain from smoking for 8 hours. Similar sampling was done for 40 smokers (acute smokers), who were asked to smoke one cigarette immediately before blood collection. Plasma was used to measure ADAMTS-13 antigen and activity levels, as well as vWF antigen and collagen binding activity levels using commercial ELISA kits. Compared to non-smokers, ADAMTS-13 and vWF activities were significantly lower in smokers at rest (p < 0.05). Acute smokers had significantly higher levels of vWF activity and ADAMTS-13 antigen and activity levels (p < 0.01), compared to smokers at rest. Our results suggest that high vWF activity is accompanied by an increase in ADAMTS-13 activity as a natural physiological mechanism to degrade the elevated vWF molecules. If not followed by a subsequent smoke, the activities of both proteins subside. It is possible that the repeated increase in vWF and constant degradation by ADAMTS-13 results in lower overall levels of both proteins in smokers (at rest) compared to nonsmokers who do not experience a similar (repeated) injury to the endothelium.

Detection of mutations in the gyrA gene in fluoroquinolone resistance Salmonella enterica serotypes typhi and paratyphi A isolated from the Infectious Diseases Hospital, Kuwait

Background: Enteric fever due to Salmonella enterica is a major health problem, and fluoroquinolones such as ciprofloxacin are mostly the antibiotic of choice for treatment. Resistance to ciprofloxacin has been noticed to increase due to the emergence of new mutations in the bacterial DNA. Aims: To explore the fluoroquinolone resistance and molecular characterisation of reduced quinolone susceptibility in S typhi and S paratyphi A in Kuwait. Methods: 136 clinical isolates of S typhi and 40 of S paratyphi A were collected over five years. The antimicrobial susceptibility was studied by various methods. DNA sequencing of gyrA, gyrB, parC and parE genes was performed in 31 isolates. Results: There was a substantial difference in MIC range between the two serotypes, with the most common MIC for S typhi being 0.25 mg/l and for S paratyphi A being 1 mg/l. The proportion of nalidixic acid resistant strains increased gradually over the years. These strains had a significantly higher range of MIC of ciprofloxacin (0.023 mg/l to 1.0 mg/l) compared to the nalidixic acid sensitive strains (0.0016 mg/l to 0.125 mg/l). DNA sequencing of gyrA gene showed the presence of three different point mutations: Ser83→Phe in 17 strains, Ser83→Leu in 3 strains and Asp87→Asn in 6 strains. No mutations in the other genes were found. Conclusions: It is very important to keep searching for new mutations and continuously monitor drug resistance in different parts of the world in order to efficiently manage cases with enteric fever.

Megaloblastosis: From Morphos to Molecules

Objective: Megaloblastosis (i.e., megaloblastic transformation of erythroid precursor cells in the bone marrow) is the cytomorphological hallmark of megaloblastic anemia resulting from vitamin B12 and folate deficiency. It is characterized by a finely stippled lacy pattern of nuclear chromatin, which is believed to be an expression of deranged cellular DNA synthesis. However, the molecular basis of these cytomorphological aberrations still remains obscure. The current presentation describes the results of our studies on some molecular events associated with the development of megaloblastosis. Methods: Transmission electron microscopy was used to study megaloblasts as well as DNA fibers extracted from megaloblastic and normoblastic bone marrows with and without treatment with proteinase K during the extraction procedure; cellular DNA synthesis in bone marrow cultures was studied by incorporation of 3H-thymidine and deoxyuridine suppression test, while histone biosynthesis in bone marrow cells was studied by in vitro incorporation of 3H-tryptophan, 3H-lysine and 3H-arginine into histones. Results: Derangement of DNA synthesis occurred due to an impaired de novo pathway of thymidylate synthesis in both vitamin-B12- and folate-deficient human megaloblastic bone marrows as well as in the bone marrows of rhesus monkeys and rats with experimentally induced folate deficiency. Interestingly, folate-deficient monkeys developed frank megaloblastic bone marrows, but folate-deficient rats did not. On the other hand, megaloblastic changes in the bone marrow of human patients with myelodysplastic syndrome and erythroleukemia were not associated with this DNA synthetic abnormality. Biosynthesis of predominantly arginine-rich histones in megaloblastic bone marrows was markedly reduced as compared to normoblastic bone marrows, which was consistently associated with elongation and despiralization of chromosomes and finely stippled nuclear chromatin in megaloblasts. Conclusion: The impaired biosynthesis of predominantly arginine-rich nuclear histones appeared to be a common molecular event (a denominator) underlying the development of megaloblastosis with or without abnormal DNA synthesis.

The emergence of carbapenem resistance in ESBL-producing Escherirchia coli O25B-ST131 strain from community acquired infection in Kuwait

In this study we investigated a multi-drug resistant E.coli recovered from ascetic fluid of a haemodialysis patient with community-onset urinary tract infection from Al-Amiri hospital in Kuwait. The patient was suffering from advanced liver disease with portal hypertension and multiple current inter abdominal abscesses.

Can we rely on one laboratory test in detection of extended-spectrum β-lactamases among Enterobacteriaceae? An evaluation of the Vitek 2 system and comparison with four other detection methods in Kuwait

Background and Aims: Clinical hospitals need to correctly identify extended spectrum β-lactamase (ESBL)-producing bacteria in infected patients to correctly treat the patient and avoid spreading antibiotic resistance. Kuwaiti hospitals use one laboratory test for detecting ESBL bacteria. This study evaluated whether that was sufficient to detect ESBL bacteria, and compared the Vitek system with other detection systems. Methods: (Klebsiella pneumoniae, Escherichia coli, K oxytoca and Enterobacter cloacae) were collected from five different Kuwaiti main hospitals, all of which were flagged as ESBL-positive by the Vitek 2 system. The isolates were retested by the Vitek 2 system, and were also tested by double disc diffusion (DDD), the disc approximation test, the E-test and the MicroScan system for the detection of ESBLs. Results: Retesting with the Vitek system revealed 100% compatibility with the results of the source hospitals. The MicroScan system, DDD, disc approximation test and E-test could detect ESBL in 199, 192, 178 and 205 isolates, respectively. Conclusions: Technically, the MicroScan and Vitek 2 systems were the least demanding method to detect ESBL as it is an integral part of the routine susceptibility test card. E-test strips were reliable but the most expensive of all techniques used. The DDD test and disc approximation, while relatively inexpensive, were technically subjective. The Vitek system may be very suitable in clinical laboratories, but would be better if accompanied with another test for detection of ESBL bacteria.

Thrombosis risk in carriers of the factor V Leiden mutation: Is it associated with a defined skin color?

Factor V Leiden (FVL; G1691A) is an autosomal dominant mutation with a high risk for thrombosis. Speculation that founders of FVL lived in the Middle East is supported by a prevalence of FVL that is higher in Arabs residing in Israel, Jordan, Lebanon, and Syria (12-14%) than in other white populations like Europeans (4-5%, up to 15% in the South of Sweden). We sought to verify the appropriate use of skin color as a clinical sign by which Arab individuals in Kuwait are included or excluded from testing for FVL. After institutional approval, 200 healthy Arabs residing in Kuwait consented to participate. Skin type was distinguished for the participants by Fitzpatrick natural skin color classification: 76 (38%) skin type II (white), 96 (48%) Mediterranean skin type IV (brown), and 28 (14%) skin type VI (black). FVL was tested by real-time PCR, and the percentage of carriers was calculated in each group. FVL was positive in 17 (8.5%) of the total subjects: 8 (10.5%) skin type II, 7 (7.3%) skin type IV, and 2 (7.1%) skin type VI. Therefore, FVL shows an even distribution in Arabs, and all Arabs residing in Kuwait should be tested for FVL irrespective of skin color.